Stabilization of poly(a) sequence encoding dna sequences

ABSTRACT

The present invention relates to nucleic acid molecules containing poly (dA:dT) regions which are stabilized in  E .- coli , methods of propagating such nucleic acid molecules in  E. coli , methods of obtaining RNA, peptides or proteins using such nucleic acid molecules and to RNA which is obtained from such nucleic acid molecules and its use. In particular, the poly (dA:dT) regions contain at least one disruption by a sequence not encoding a sequence solely composed of A residues.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/325,280 filed Jan. 10, 2017, which is a National Stage Entry patentapplication of PCT/EP2015/065357, filed on Jul. 6, 2015, which claimsforeign priority to International Patent Application No.PCT/EP2014/064924, filed on Jul. 11, 2014, the disclosures of each whichare hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 9, 2020, isnamed 9767751_1.txt and is 855 bytes in size.

BACKGROUND

The use of RNA offers an attractive alternative to DNA in order tocircumvent the potential safety risks connected with the therapeutic useof DNA. In vitro-transcribed RNA (IVT-RNA) is of particular interest intherapeutic approaches. The advantages of a therapeutic use of RNAinclude transient expression and a non-transforming character. RNA doesnot need to enter the nucleus in order to be expressed and moreovercannot integrate into the host genome, thereby eliminating the risk ofoncogenesis. When used for vaccination, injection of RNA can induce bothcellular and humoral immune responses in vivo. However, the use of RNAfor clinical applications is greatly restricted especially by the shorthalf life of RNA.

IVT vectors may be used in a standardized manner as template for invitro transcription. Such IVT vectors may have the following structure:a 5′ RNA polymerase promoter enabling RNA transcription, followed by agene of interest which is flanked either 3′ and/or 5′ by untranslatedregions (UTR), and a 3′ polyadenyl cassette containing A nucleotides.Prior to in vitro transcription, the circular plasmid is linearizeddownstream of the polyadenyl cassette by type II restriction enzymes(recognition sequence corresponds to cleavage site). The polyadenylcassette thus corresponds to the later poly(A) sequence in thetranscript.

The 3′ poly(A) sequence of RNA is important for nuclear export, RNAstability and translational efficiency of eukaryotic messenger RNA(mRNA). The 3′ poly(A) sequence is shortened over time and if shortenough, the RNA is degraded enzymatically.

We have previously demonstrated that a 3′ poly(A) sequence with a lengthof 120 nucleotides (A120) has a predominant effect on RNA stability andtranslational efficiency and thus, is beneficial for all-over RNAefficacy.

However, it has been observed that the DNA sequence encoding the 3′poly(A) sequence (3′ polyadenyl cassette), i.e. a stretch of consecutivedA:dT base pairs, is subjected to shortening in some bacterialsubclones, when propagated in E. coli. Accordingly, before producing theplasmid DNA as the starting material for in vitro transcription a largenumber of bacterial clones has to be tested, e.g., by determining thelength of the 3′ polyadenyl cassette by suitable restriction analysis,to obtain a single clone with a 3′ polyadenyl cassette of the correctlength encoding a 3′ poly(A) sequence of the correct length.

It was the object of the present invention to find a 3′ polyadenylcassette which shows constant propagation with the coding plasmid DNA inE. coli and which encodes a 3′ poly(A) sequence maintaining the effectswith respect to supporting RNA stability and translational efficiency.

This object is achieved according to the invention by the subject matterof the claims.

According to the invention, it was found that a disruption of the 3′polyadenyl cassette (poly(dA:dT) region) by a 10 nucleotide randomsequence, with an equal distribution of the 4 nucleotides (linker), hasonly minor influence on functionality of the encoded RNA but increasesthe stability of the 3′ polyadenyl cassette in E. coli. Further, neitherthe sequence nor the position of the linker within the 3′ poly(A)sequence resulted in a reduction of translational efficiency andstability of the in vitro transcribed RNA (IVT RNA).

For stability testing of the IVT vector region encoding the 3′ poly(A)sequence, the SIINFEKL peptide (SEQ ID NO: 1) was cloned upstream of thepoly(dA:dT) region.

This construct showed a poly(dA:dT) instability (i.e. percentage ofclones upon propagation with shortened poly(dA:dT)) of 50-60%. Detailedanalysis, using the described restriction analysis method identified theregion at position 30-50 as being particular sensitive to shortening ofthe poly(dA:dT) stretch. Introduction of a 10 nucleotide random sequencein this sensitive region led to an increase of the poly(dA:dT)stability. Constructs with 30 or 40 adenosine nucleotides, followed bythe linker sequence and another 70 or 60 adenosines (A30L70 and A40L60)respectively, resulted in an poly(dA:dT) instability of only 3-4% in E.coli. Results were confirmed by testing the constructs in severaldifferent E. coli strains.

Functionality of IVT RNA encoded by DNA carrying the stabilizedpoly(dA:dT)-tails was tested in different assays. Electroporation of theIVT RNA in somatic cell lines but also in immune cells such as immaturedendritic cells showed no difference in translational capacity comparedto the A120 over a time period of 72 hours. Injection of luciferaseencoding IVT RNA into mice confirmed an equal protein translationindependent of the inserted type of the 3′ poly(A) sequence.

An impact on the immunological response of the different 3′ poly(A)sequences was analyzed by comparison of the amount of antigen-specificCD8⁺ T-cells upon injection of SIINFEKL (SEQ ID NO: 1) IVT RNA. Theexperiments revealed no difference between the A120 and its stabilizedversions A30L70 and A40L60.

Taken together, we show that the insertion of a 10 nucleotide randomsequence between position 30 and 50 of a poly(dA:dT) region results in amore than 10-fold sequence stabilization in E. coli. The correspondingmodified 3′ poly(A) sequence of the RNA transcribed from the templateDNA has the same functionality, i.e. stability and translationalefficiency in vivo and in vitro as the classical A120. Additionally theimmunological response is not altered by the use of a modified poly(A)sequence.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a nucleic acid moleculecomprising in the 5′→3′ direction of transcription:

(a) a promoter;

(b) a transcribable nucleic acid sequence or a nucleic acid sequence forintroducing a transcribable nucleic acid sequence; and

(c) a nucleic acid sequence which, when transcribed under the control ofthe promoter (a), codes for a nucleotide sequence of at least 80consecutive nucleotides in the transcript, wherein said nucleotidesequence of at least 80 consecutive nucleotides in the transcript is apolyadenyl sequence comprising within the polyadenyl sequence a sequenceof one or more consecutive nucleotides containing nucleotides other thanA nucleotides.

In one embodiment, said sequence of one or more consecutive nucleotidescontaining nucleotides other than A nucleotides is a sequence,preferably an arbitrary sequence, of 2 or more consecutive nucleotides,wherein the first and the last nucleotide of said sequence of 2 or moreconsecutive nucleotides is a nucleotide other than an A nucleotide.

In other words, the nucleic acid molecule of the invention contains 3′polyadenyl cassette (poly(dA:dT) region) containing at least onedisruption by a sequence not encoding a sequence solely composed of Aresidues, i.e. the poly(dA:dT) region is disrupted by one or morestretches of basepairs comprising basepairs other than (dA:dT). Thus,the nucleic acid sequence (c), when transcribed under the control of thepromoter (a), codes for a nucleotide sequence of at least 80 consecutivenucleotides in the transcript, wherein said nucleotide sequence of atleast 80 consecutive nucleotides in the transcript is a polyadenylsequence wherein at least one portion of the polyadenyl sequence isreplaced by a sequence containing nucleotides other than A nucleotidessuch as a sequence of 2 or more consecutive nucleotides, wherein thefirst and the last nucleotide of said sequence of 2 or more nucleotidesis a nucleotide other than an A nucleotide. In other words, the nucleicacid sequence (c), when transcribed under the control of the promoter(a), codes for a polyadenyl sequence containing interspersed within saidpolyadenyl sequence one or more sequence stretches of one or morenucleotides, wherein said sequence stretches each are not an Anucleotide or a stretch of A nucleotides, i.e. an oligo-A sequence or apoly-A sequence.

In one embodiment, said nucleic acid molecule is a DNA molecule. In oneembodiment, said nucleic acid molecule is an expression vector orplasmid such as an IVT vector.

In one embodiment, said nucleic acid sequence (c) exhibits higherstability upon propagation of said nucleic acid molecule in E. colicompared to a nucleic acid molecule which comprises instead of saidnucleic acid sequence (c) a nucleic acid sequence (c)′ which, whentranscribed under the control of the promoter (a), codes for apolyadenyl sequence of the same length as said nucleotide sequence of atleast 80 consecutive nucleotides in the transcript.

In one embodiment, said nucleotide sequence of at least consecutivenucleotides comprises at least 90 nucleotides, preferably at least 100nucleotides, preferably at least 110 nucleotides. In one embodiment,said nucleotide sequence of at least 80 consecutive nucleotidescomprises about 120 nucleotides. In particular embodiments, saidnucleotide sequence of at least 80 consecutive nucleotides comprises upto 200, preferably up to 150, and, in particular, up to 130 nucleotides.In one embodiment, at least 90%, preferably at least 92%, preferably atleast 95%, 97% or 98% of the nucleotides of said nucleotide sequence ofat least 80 consecutive nucleotides are A nucleotides in said polyadenylsequence (not including A nucleotides in said sequence of one or moreconsecutive nucleotides containing nucleotides other than Anucleotides).

In one embodiment, said sequence of one or more consecutive nucleotidescontaining nucleotides other than A nucleotides is located within aregion from position 21 to position 80, preferably from position 21 toposition 60, more preferably from position 31 to position 50 of saidpolyadenyl sequence.

In one embodiment, said sequence of one or more consecutive nucleotidescontaining nucleotides other than A nucleotides is preceeded by at least20 A residues, preferably at least 30, 40 or 50 A residues in saidpolyadenyl sequence. In particular embodiments, said sequence of one ormore consecutive nucleotides containing nucleotides other than Anucleotides is preceeded by up to 80 A residues, preferably up to 70 or60 A residues in said polyadenyl sequence.

In one embodiment, said sequence of one or more consecutive nucleotidescontaining nucleotides other than A nucleotides is followed by at least20 A residues, preferably at least 30, 40, 50, 60 or 70 A residues insaid polyadenyl sequence. In particular embodiments, said sequence ofone or more consecutive nucleotides containing nucleotides other than Anucleotides is followed by up to 100 A residues, preferably up to 80 Aresidues in said polyadenyl sequence.

In one embodiment, said sequence of one or more consecutive nucleotidescontaining nucleotides other than A nucleotides is preceeded by 20 to50, preferably 30 to 40 A residues in said polyadenyl sequence and isfollowed by 30 to 80, preferably 40 to 70 A residues in said polyadenylsequence.

In one embodiment, said sequence of one or more consecutive nucleotidescontaining nucleotides other than A nucleotides has a length of at least3, at least 4, at least 5, at least 6, at least 8, preferably at least10, more preferably at least 15 nucleotides.

In one embodiment, said sequence of one or more consecutive nucleotidescontaining nucleotides other than A nucleotides has a length of no morethan 50, preferably no more than 30, more preferably no more than 20nucleotides.

In one embodiment, said sequence of one or more consecutive nucleotidescontaining nucleotides other than A nucleotides does not comprise morethan 3, preferably no more than 2, preferably no consecutive A residues.

In one embodiment, the nucleic acid sequences (b) and (c) under thecontrol of the promoter (a) can be transcribed to give a commontranscript.

In one embodiment, in the transcript said nucleotide sequence of atleast 80 consecutive nucleotides is located at the 3′ end.

In one embodiment, the nucleic acid molecule of the invention is aclosed circular molecule or a linear molecule.

In one embodiment, the transcribable nucleic acid sequence comprises anucleic acid sequence coding for a peptide or protein and the nucleicacid sequence for introducing a transcribable nucleic acid sequence is amultiple cloning site.

In one embodiment, the nucleic acid molecule of the invention furthercomprises one or more members selected from the group consisting of: (i)a reporter gene; (ii) a selectable marker; and (iii) an origin ofreplication.

In one embodiment, the nucleic acid molecule of the invention issuitable, in particular after linearization, for in vitro transcriptionof RNA, in particular mRNA.

Preferably, the nucleic acid sequence transcribed from the nucleic acidsequence (c), i.e., said nucleotide sequence of at least 80 consecutivenucleotides, is preferably active so as to increase the translationefficiency and/or the stability of the nucleic acid sequence transcribedfrom the transcribable nucleic acid sequence (b).

Prior to in vitro transcription, circular IVT vectors are generallylinearized downstream of the polyadenyl cassette by type II restrictionenzymes (recognition sequence corresponds to cleavage site). Thepolyadenyl cassette thus corresponds to the later poly(A) sequence inthe transcript. As a result of this procedure, some nucleotides remainas part of the enzyme cleavage site after linearization and extend ormask the poly(A) sequence at the 3′ end. However, it was found that RNAhaving an open-ended poly(A) sequence is translated more efficientlythan RNA having a poly(A) sequence with a masked terminus.

Accordingly, nucleic acid molecules of the invention when used asexpression vectors preferably allow transcription of RNA with a poly(A)sequence which preferably has an open end in said RNA, i.e. nonucleotides other than A nucleotides flank said poly(A) sequence at its3′ end. An open-ended poly(A) sequence in the RNA can be achieved byintroducing a type IIS restriction cleavage site into an expressionvector which allows RNA to be transcribed under the control of a 5′ RNApolymerase promoter and which contains a polyadenyl cassette, whereinthe recognition sequence is located downstream of the polyadenylcassette, while the cleavage site is located upstream and thus withinthe polyadenyl cassette. Restriction cleavage at the type IISrestriction cleavage site enables a plasmid to be linearized within thepolyadenyl cassette. The linearized plasmid can then be used as templatefor in vitro transcription, the resulting transcript ending in anunmasked poly(A) sequence.

Accordingly, in one embodiment, it is preferred that the nucleic acidmolecule of the invention can be cleaved, preferably enzymatically or inanother biochemical way, within the nucleic acid sequence (c) in such away that said cleavage results in a nucleic acid molecule whichcomprises, in the 5′→3′ direction of transcription, the promoter (a),the nucleic acid sequence (b), and at least a part of the nucleic acidsequence (c), wherein the at least a part of the nucleic acid sequence(c), when transcribed under the control of the promoter (a), codes forsaid nucleotide sequence of at least 80 consecutive nucleotides in thetranscript and wherein in the transcript the 3′-terminal nucleotide isan A nucleotide of said nucleotide sequence of at least 80 consecutivenucleotides.

Preferably, after cleavage, the nucleic acid molecule, at the end of thestrand that serves as template for the nucleotide sequence of at least80 consecutive nucleotides, has a T nucleotide which is part of thenucleic acid sequence which serves as template for the nucleotidesequence of at least 80 consecutive nucleotides in the transcript.

The nucleic acid molecule of the invention is preferably a closedcircular molecule prior to cleavage and a linear molecule aftercleavage.

Preferably, cleavage is carried out with the aid of a restrictioncleavage site which is preferably a restriction cleavage site for a typeIIS restriction endonuclease.

In one embodiment, the recognition sequence for the type IIS restrictionendonuclease is located 5-26 base pairs, preferably 24-26 base pairs,downstream of the 3′ end of the nucleic acid sequence (c).

In one embodiment, a nucleic acid molecule according to the invention isin a closed circular conformation and preferably suitable for in vitrotranscription of RNA, in particular mRNA, in particular afterlinearization.

In further aspects, the invention relates to a nucleic acid moleculeobtainable by linearization of an above-described nucleic acid molecule,preferably by cleavage within the nucleic acid sequence (c), and to RNAobtainable by transcription, preferably in vitro transcription, withabove-described nucleic acid molecules under the control of the promoter(a).

In a further aspect, the invention relates to a method of propagating anucleic acid molecule, comprising:

-   -   (i) providing a nucleic acid molecule of the invention, and    -   (ii) propagating said nucleic acid molecule in E. coli.

In one embodiment, propagating said nucleic acid molecule in E. colicomprises transforming E. coli with said nucleic acid molecule andcultivating said transformed E. coli.

In one embodiment, the method of the invention further comprisesisolating said nucleic acid molecule from E. coli following propagation.

In a further aspect, the invention relates to a method of obtaining RNA,comprising:

-   -   (i) propagating a nucleic acid molecule according to a method of        the invention of propagating a nucleic acid molecule, and    -   (ii) transcribing in vitro RNA using the nucleic acid molecule        as a template.

In a further aspect, the invention relates to a method of obtaining apeptide or protein, comprising:

-   -   (i) obtaining mRNA encoding the peptide or protein according to        a method of the invention of obtaining RNA, and    -   (ii) translating the mRNA.

In one embodiment, the method of obtaining RNA or the method ofobtaining a peptide or protein further comprises, prior to transcriptionof the nucleic acid molecule, cleavage of the nucleic acid molecule.

In one embodiment, cleavage is within the nucleic acid sequence (c) insuch a way that transcription of the nucleic acid obtained in this waygenerates a transcript which has at its 3′-terminal end said nucleotidesequence of at least 80 consecutive nucleotides, wherein the 3′-terminalnucleotide of said transcript is an A nucleotide of the nucleotidesequence of at least 80 consecutive nucleotides.

In all aspects of the methods according to the invention, cleavage ispreferably carried out with the aid of a restriction cleavage site whichis preferably a restriction cleavage site for a type IIS restrictionendonuclease.

In one embodiment, the recognition sequence for the type IIS restrictionendonuclease is 5-26 base pairs, preferably 24-26 base pairs, downstreamof the 3′ end of the nucleic acid sequence (c).

The invention also relates to RNA obtainable by the methods according tothe invention of obtaining RNA.

The invention may be utilized, for example, for increasing expression ofrecombinant proteins in cellular transcription and expression. Morespecifically, it is possible, when producing recombinant proteins, touse expression vectors of the invention for transcription of recombinantnucleic acids and expression of recombinant proteins in cell-basedsystems. This includes, for example, the preparation of recombinantantibodies, hormones, cytokines, enzymes, and the like. This allowsinter alia production costs to be reduced.

It is also possible to use the nucleic acid molecules of the inventionfor gene therapy applications. Accordingly, a nucleic acid molecule ofthe invention may be a gene therapy vector and used for expression of atransgene. To this end, any nucleic acid (DNA/RNA)-based vector systems(for example plasmids, adenoviruses, poxvirus vectors, influenza virusvectors, alphavirus vectors, and the like) may be used. Cells can betransfected with these vectors in vitro, for example in lymphocytes ordendritic cells, or else in vivo by direct administration.

RNA of the invention (obtained using a nucleic acid molecule describedherein as a transcription template) may be employed, for example, fortransient expression of genes, with possible fields of application beingRNA-based vaccines which are transfected into cells in vitro oradministered directly in vivo, transient expression of functionalrecombinant proteins in vitro, for example in order to initiatedifferentiation processes in cells or to study functions of proteins,and transient expression of functional recombinant proteins such aserythropoietin, hormones, coagulation inhibitors, etc., in vivo, inparticular as pharmaceuticals.

RNA of the invention may be used in particular for transfectingantigen-presenting cells and thus as a tool for delivering the antigento be presented and for loading antigen-presenting cells, with saidantigen to be presented corresponding to the peptide or proteinexpressed from said RNA or being derived therefrom, in particular by wayof intracellular processing such as cleavage, i.e. the antigen to bepresented is, for example, a fragment of the peptide or proteinexpressed from the RNA. Such antigen-presenting cells may be used forstimulating T cells, in particular CD4⁺ and/or CD8⁺ T cells.

Accordingly, in a further aspect, the invention relates to a use of theRNA of the invention for transfecting a host cell. In one embodiment,the host cell is an antigen-presenting cell, in particular a dendriticcell, a monocyte or a macrophage.

In a further aspect, the invention relates to a use of the RNA of theinvention for vaccination.

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise. For example, if in a preferredembodiment a sequence of one or more consecutive nucleotides containingnucleotides other than A nucleotides is preceeded by at least 20 Aresidues in said polyadenyl sequence and if in another preferredembodiment a sequence of one or more consecutive nucleotides containingnucleotides other than A nucleotides is followed by at least 20 Aresidues in said polyadenyl sequence, it is a contemplated preferredembodiment that a sequence of one or more consecutive nucleotidescontaining nucleotides other than A nucleotides is preceeded andfollowed by at least 20 A residues in said polyadenyl sequence.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kolbl, Eds.,Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, cellbiology, immunology, and recombinant DNA techniques which are explainedin the literature in the field (cf., e.g., Molecular Cloning: ALaboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold SpringHarbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps. The terms “a” and “an” and “the”and similar reference used in the context of describing the invention(especially in the context of the claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. Recitation of ranges of values hereinis merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range. Unlessotherwise indicated herein, each individual value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”),provided herein is intended merely to better illustrate the inventionand does not pose a limitation on the scope of the invention otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of theinvention.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

The present invention describes nucleic acid molecules such as DNAplasmids useful as RNA expression vectors comprising a modified 3′poly(dA:dT) cassette (encoding a modified 3′ poly(A) sequence) whichshows constant propagation without being subject to shortening in E.coli.

E. coli is a gram-negative, facultatively anaerobic, rod-shapedbacterium of the genus Escherichia that is commonly found in the lowerintestine of warm-blooded organisms. The bacterium can be grown easilyand inexpensively in a laboratory setting, and has been intensivelyinvestigated for over 60 years. E. coli is the most widely studiedprokaryotic model organism, and an important species in the fields ofbiotechnology and microbiology, where it has served as the host organismfor the majority of work with recombinant DNA. E. coli strains accordingto the invention include: AG1, AB1157, B2155, BL21, BNN93, BNN97,BW26434, C600, CSH50, D1210, DB3.1, DH1, DH5a, DH10B, DH12S, DM1, E.cloni(r), E. coli K12 ER2738, ER2566, ER2267, HB101, IJ1126, IJ1127,JM83, JM101, JM103, JM105, JM106, JM107, JM108, JM109, JM110, JM2.300,LE392, Mach1, MC1061, MC4100, MFDpir, MG1655, OmniMAX2, RR1, RV308,SOLR, SS320, STBL2, STBL3, STBL4, SURE, SURE2, TG1, TOP10, Top10F′,W3110, WM3064, XL1-Blue, XL2-Blue, XL1-Red and XL10-Gold.

According to the invention, a nucleic acid molecule or a nucleic acidsequence refers to a nucleic acid which is preferably deoxyribonucleicacid (DNA) or ribonucleic acid (RNA). According to the invention,nucleic acids comprise genomic DNA, cDNA, mRNA, recombinantly preparedand chemically synthesized molecules. According to the invention, anucleic acid may be in the form of a single-stranded or double-strandedand linear or covalently closed circular molecule.

In the context of the present invention, the term “RNA” relates to amolecule which comprises ribonucleotide residues and preferably beingentirely or substantially composed of ribonucleotide residues. The term“ribonucleotide” relates to a nucleotide with a hydroxyl group at the2′-position of a β-D-ribofuranosylgroup. The term “RNA” comprisesdouble-stranded RNA, single stranded RNA, isolated RNA such as partiallyor completely purified RNA, essentially pure RNA, synthetic RNA, andrecombinantly generated RNA such as modified RNA which differs fromnaturally occurring RNA by addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of a RNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in RNA molecules can also comprise non-standard nucleotides,such as non-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs, particularly analogs of naturally-occurring RNAs. Accordingto the invention, RNA includes mRNA.

The term “mRNA” means “messenger-RNA” and relates to a transcript whichis generated by using a DNA template and encodes a peptide or protein.Typically, mRNA comprises a 5′-UTR, a protein coding region, and a3′-UTR. mRNA may be generated by in vitro transcription from a DNAtemplate. The in vitro transcription methodology is known to the skilledperson. For example, there is a variety of in vitro transcription kitscommercially available. According to the invention, mRNA may be modifiedby further stabilizing modifications and capping, in addition to themodifications according to the invention.

In one embodiment, the term “modification” relates to providing a RNAwith a 5′-cap or 5′-cap analog. The term “5′-cap” refers to a capstructure found on the 5′-end of an mRNA molecule and generally consistsof a guanosine nucleotide connected to the mRNA via an unusual 5′ to 5′triphosphate linkage. In one embodiment, this guanosine is methylated atthe 7-position. The term “conventional 5′-cap” refers to a naturallyoccurring RNA 5′-cap, preferably to the 7-methylguanosine cap (m⁷G). Inthe context of the present invention, the term “5′-cap” includes a5′-cap analog that resembles the RNA cap structure and is modified topossess the ability to stabilize RNA if attached thereto, preferably invivo and/or in a cell. Providing an RNA with a 5′-cap or 5′-cap analogmay be achieved by in vitro transcription of a DNA template in thepresence of said 5′-cap or 5′-cap analog, wherein said 5′-cap isco-transcriptionally incorporated into the generated RNA strand, or theRNA may be generated, for example, by in vitro transcription, and the5′-cap may be generated post-transcriptionally using capping enzymes,for example, capping enzymes of vaccinia virus.

The term “nucleic acid” according to the invention also comprises achemical derivatization of a nucleic acid on a nucleotide base, on thesugar or on the phosphate, and nucleic acids containing non-naturalnucleotides and nucleotide analogs.

According to the invention, a “nucleic acid sequence which is derivedfrom a nucleic acid sequence” refers to a nucleic acid containing, incomparison with the nucleic acid from which it is derived, single ormultiple nucleotide substitutions, deletions and/or additions.Preferably there is a certain degree of homology between said nucleicacids and the nucleotide sequences of said nucleic acids correspond in asignificant direct or complementary manner. According to the invention,a nucleic acid derived from a nucleic acid has a functional property ofthe nucleic acid from which it is derived. Such functional propertiesinclude in particular the ability to increase, in a functional linkageto a nucleic acid which can be transcribed into RNA (transcribablenucleic acid sequence), the stability and/or translation efficiency ofRNA produced from this nucleic acid in the complete RNA molecule.

According to the invention, “functional linkage” or “functionallylinked” relates to a connection within a functional relationship. Anucleic acid is “functionally linked” if it is functionally related toanother nucleic acid sequence. For example, a promoter is functionallylinked to a coding sequence if it influences transcription of saidcoding sequence.

Functionally linked nucleic acids are typically adjacent to one another,where appropriate separated by further nucleic acid sequences, and, inparticular embodiments, are transcribed by RNA polymerase to give asingle RNA molecule (common transcript).

The nucleic acids described according to the invention are preferablyisolated. The term “isolated nucleic acid” means according to theinvention that the nucleic acid has been (i) amplified in vitro, forexample by polymerase chain reaction (PCR), (ii) recombinantly producedby cloning, (iii) purified, for example by cleavage andgel-electrophoretic fractionation, or (iv) synthesized, for example bychemical synthesis. An isolated nucleic acid is a nucleic acid availableto manipulation by recombinant DNA techniques.

A nucleic acid is “complementary” to another nucleic acid if the twosequences can hybridize with one another and form a stable duplex, saidhybridization being carried out preferably under conditions which allowspecific hybridization between polynucleotides (stringent conditions).Stringent conditions are described, for example, in Molecular Cloning: ALaboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. 1989 or CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., John Wiley &Sons, Inc., New York, and refer, for example, to a hybridization at 65°C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02%polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH₂PO₄ (pH7),0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodiumcitrate, pH 7. After hybridization, the membrane to which the DNA hasbeen transferred, is washed, for example, in 2×SSC at room temperatureand then in 0.1-0.5×SSC/0.1×SDS at temperatures up to 68° C.

According to the invention, homologous nucleic acids have nucleotideswhich are at least 60%, at least 70%, at least 80%, at least 90%, andpreferably at least 95%, at least 98% or at least 99%, identical.

The term “% identical” is intended to refer to a percentage ofnucleotides which are identical in an optimal alignment between twosequences to be compared, with said percentage being purely statistical,and the differences between the two sequences may be randomlydistributed over the entire length of the sequence and the sequence tobe compared may comprise additions or deletions in comparison with thereference sequence, in order to obtain optimal alignment between twosequences. Comparisons of two sequences are usually carried out bycomparing said sequences, after optimal alignment, with respect to asegment or “window of comparison”, in order to identify local regions ofcorresponding sequences. The optimal alignment for a comparison may becarried out manually or with the aid of the local homology algorithm bySmith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of thelocal homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol.48, 443, and with the aid of the similarity search algorithm by Pearsonand Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444 or with the aid ofcomputer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P,BLAST N and TFASTA in Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Drive, Madison, Wis.).

Percentage identity is obtained by determining the number of identicalpositions in which the sequences to be compared correspond, dividingthis number by the number of positions compared and multiplying thisresult by 100.

For example, the BLAST program “BLAST 2 sequences” which is available onthe website http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi may beused.

“3′ end of a nucleic acid” refers according to the invention to that endwhich has a free hydroxy group.

In a diagrammatic representation of double-stranded nucleic acids, inparticular DNA, the 3′ end is always on the right-hand side. “5′ end ofa nucleic acid” refers according to the invention to that end which hasa free phosphate group. In a diagrammatic representation ofdouble-strand nucleic acids, in particular DNA, the 5′ end is always onthe left-hand side.

5′ end 5′--P-NNNNNNN-OH-3′ 3′ end 3′-HO-NNNNNNN-P--5′

In particular embodiments, a nucleic acid is functionally linkedaccording to the invention to expression control sequences which may behomologous or heterologous with respect to the nucleic acid.

A transcribable nucleic acid sequence, in particular a nucleic acidsequence coding for a peptide or protein, and an expression controlsequence are “functionally” linked to one another, if they arecovalently linked to one another in such a way that transcription orexpression of the transcribable and in particular coding nucleic acidsequence is under the control or under the influence of the expressioncontrol sequence.

If the nucleic acid sequence is to be translated into a functionalpeptide or protein, induction of an expression control sequencefunctionally linked to the coding sequence results in transcription ofsaid coding sequence, without causing a frame shift in the codingsequence or the coding sequence being unable to be translated into thedesired peptide or protein.

The term “expression control sequence” comprises according to theinvention promoters, ribosome-binding sequences and other controlelements which control transcription of a gene or translation of thederived RNA. In particular embodiments of the invention, the expressioncontrol sequences can be regulated. The precise structure of expressioncontrol sequences may vary depending on the species or cell type butusually includes 5′-untranscribed and 5′- and 3′-untranslated sequencesinvolved in initiating transcription and translation, respectively, suchas TATA box, capping sequence, CAAT sequence and the like. Morespecifically, 5′-untranscribed expression control sequences include apromoter region which encompasses a promoter sequence for transcriptioncontrol of the functionally linked gene. Expression control sequencesmay also include enhancer sequences or upstream activator sequences.

The nucleic acid sequences specified herein, in particular transcribableand coding nucleic acid sequences, may be combined with any expressioncontrol sequences, in particular promoters, which may be homologous orheterologous to said nucleic acid sequences, with the term “homologous”referring to the fact that a nucleic acid sequence is also functionallylinked naturally to the expression control sequence, and the term“heterologous” referring to the fact that a nucleic acid sequence is notnaturally functionally linked to the expression control sequence.

The term “promoter” or “promoter region” refers to a DNA sequenceupstream (5′) of the coding sequence of a gene, which controlsexpression of said coding sequence by providing a recognition andbinding site for RNA polymerase. The promoter region may include furtherrecognition or binding sites for further factors involved in regulatingtranscription of said gene. A promoter may control transcription of aprokaryotic or eukaryotic gene. A promoter may be “inducible” andinitiate transcription in response to an inducer, or may be“constitutive” if transcription is not controlled by an inducer. Aninducible promoter is expressed only to a very small extent or not atall, if an inducer is absent. In the presence of the inducer, the geneis “switched on” or the level of transcription is increased. This isusually mediated by binding of a specific transcription factor.

Examples of promoters preferred according to the invention are promotersfor SP6, T3 or T7 polymerase.

According to the invention, the term “expression” is used in its mostgeneral meaning and comprises production of RNA or of RNA and protein.It also comprises partial expression of nucleic acids. Furthermore,expression may be transient or stable. With respect to RNA, the term“expression” or “translation” relates to the process in the ribosomes ofa cell by which a strand of messenger RNA directs the assembly of asequence of amino acids to make a peptide or protein.

The term “nucleic acid sequences which can be transcribed to give acommon transcript” means that said nucleic acid sequences arefunctionally linked to one another in such a way that, where appropriateafter linearization such as restriction enzyme cleavage of the nucleicacid molecule comprising said nucleic acid sequences, in particular of aclosed circular nucleic acid molecule, transcription under the controlof a promoter results in an RNA molecule comprising the transcripts ofsaid nucleic acid sequences covalently bound to one another, whereappropriate separated by sequences located inbetween.

In the context of the present invention, the term “transcription”relates to a process, wherein the genetic code in a DNA sequence istranscribed into RNA. Subsequently, the RNA may be translated intoprotein. According to the present invention, the term “transcription”comprises “in vitro transcription”, wherein the term “in vitrotranscription” relates to a process wherein RNA, in particular mRNA, isin vitro synthesized in a cell-free system. Preferably, cloning vectorsare applied for the generation of transcripts. These cloning vectors aregenerally designated as transcription vectors and are according to thepresent invention encompassed by the term “vector”. According to thepresent invention, RNA preferably is in vitro transcribed RNA (IVT-RNA)and may be obtained by in vitro transcription of an appropriate DNAtemplate. The promoter for controlling transcription can be any promoterfor any RNA polymerase. A DNA template for in vitro transcription may beobtained by cloning of a nucleic acid, in particular cDNA, andintroducing it into an appropriate vector for in vitro transcription.The cDNA may be obtained by reverse transcription of RNA.

The term “nucleic acid sequence transcribed from a nucleic acidsequence” refers to RNA, where appropriate as part of a complete RNAmolecule, which is a transcription product of the latter nucleic acidsequence.

The term “nucleic acid sequence which is active in order to increase thetranslation efficiency and/or stability of a nucleic acid sequence”means that the first nucleic acid sequence is capable of modifying, in acommon transcript with the second nucleic acid sequence, the translationefficiency and/or stability of said second nucleic acid sequence in sucha way that said translation efficiency and/or stability is increased incomparison with the translation efficiency and/or stability of saidsecond nucleic acid sequence without said first nucleic acid sequence.In this context, the term “translation efficiency” relates to the amountof translation product provided by an RNA molecule within a particularperiod of time and the term “stability” relates to the half life of anRNA molecule.

It has been demonstrated that a double 3′-untranslated region (UTR), inparticular of the human beta-globin gene, in an RNA molecule improvestranslation efficiency in a way which clearly exceeds the total effectto be expected using two individual UTRs.

Modification, and thereby stabilization and/or increase in translationefficiency, of RNA can be achieved according to the invention bygenetically modifying expression nucleic acid molecules of the inventionwhen used as expression vectors in such a way that they allowtranscription of RNA with two or more 3′-untranslated regions at its 3′end, and preferably between the sequence coding for a peptide or protein(open reading frame) and the poly(A) sequence or the poly(A) sequencecomprising a sequence of one or more consecutive nucleotides containingnucleotides other than A nucleotides.

Accordingly, a nucleic acid molecule of the invention may comprise,preferably between the nucleic acid sequence (b) and the nucleic acidsequence (c) two or more nucleic acid sequences, each of said two ormore nucleic acid sequences corresponding to the 3′-untranslated regionof a gene or being derived therefrom. Said two or more nucleic acidsequences may be identical or different. In preferred embodiments, saidtwo or more nucleic acid sequences are independently of one anotherderived from a gene selected from the group consisting of globin genessuch as alpha2-globin, alpha1-globin, beta-globin and growth hormone,preferably human beta-globin.

The 3′-untranslated region relates to a region which is located at the3′ end of a gene, downstream of the termination codon of aprotein-encoding region, and which is transcribed but is not translatedinto an amino acid sequence.

According to the invention, a first polynucleotide region is consideredto be located downstream of a second polynucleotide region, if the 5′end of said first polynucleotide region is the part of said firstpolynucleotide region closest to the 3′ end of said secondpolynucleotide region.

The 3′-untranslated region typically extends from the termination codonfor a translation product to the poly(A) sequence which is usuallyattached after the transcription process. The 3′-untranslated regions ofmammalian mRNA typically have a homology region known as the AAUAAAhexanucleotide sequence. This sequence is presumably the poly(A)attachment signal and is frequently located from 10 to 30 bases upstreamof the poly(A) attachment site.

3′-untranslated regions may contain one or more inverted repeats whichcan fold to give stem-loop structures which act as barriers forexoribonucleases or interact with proteins known to increase RNAstability (e.g. RNA-binding proteins).

5′- and/or 3′-untranslated regions may, according to the invention, befunctionally linked to a transcribable and in particular coding nucleicacid, so as for these regions to be associated with the nucleic acid insuch a way that the stability and/or translation efficiency of the RNAtranscribed from said transcribable nucleic acid are increased.

The 3′-untranslated regions of immunoglobulin mRNAs are relatively short(fewer than about 300 nucleotides), while the 3′-untranslated regions ofother genes are relatively long. For example, the 3′-untranslated regionof tPA is about 800 nucleotides in length, that of factor VIII is about1800 nucleotides in length and that of erythropoietin is about 560nucleotides in length.

It can be determined according to the invention, whether a3′-untranslated region or a nucleic acid sequence derived therefromincreases the stability and/or translation efficiency of RNA, byincorporating the 3′-untranslated region or the nucleic acid sequencederived therefrom into the 3′-untranslated region of a gene andmeasuring whether said incorporation increases the amount of proteinsynthesized.

The above applies accordingly to the case in which according to theinvention a nucleic acid comprises two or more 3′-untranslated regionswhich are preferably coupled sequentially with or without a linkerinbetween, preferably in a “head-to-tail relationship” (i.e. the3′-untranslated regions have the same orientation, preferably theorientation naturally occurring in a nucleic acid).

According to the invention, the term “gene” refers to a particularnucleic acid sequence which is responsible for producing one or morecellular products and/or for achieving one or more intercellular orintracellular functions. More specifically, said term relates to a DNAsection which comprises a nucleic acid coding for a specific protein ora functional or structural RNA molecule.

Polyadenylation is the addition of a poly(A) sequence or tail to aprimary transcript RNA. The poly(A) sequence consists of multipleadenosine monophosphates. In other words, it is a stretch of RNA thathas only adenine bases. In eukaryotes, polyadenylation is part of theprocess that produces mature messenger RNA (mRNA) for translation. It,therefore, forms part of the larger process of gene expression. Theprocess of polyadenylation begins as the transcription of a genefinishes, or terminates. The 3′-most segment of the newly made pre-mRNAis first cleaved off by a set of proteins; these proteins thensynthesize the poly(A) sequence at the RNA's 3′ end. The poly(A)sequence is important for the nuclear export, translation, and stabilityof mRNA. The sequence is shortened over time, and, when it is shortenough, the mRNA is enzymatically degraded.

The terms “polyadenyl sequence”, “poly(A) sequence” or “poly(A) tail”refer to a sequence of adenyl residues which is typically located at the3′ end of an RNA molecule. The invention provides for such a sequence tobe attached during RNA transcription by way of a DNA template on thebasis of repeated thymidyl residues in the strand complementary to thecoding strand, whereas said sequence is normally not encoded in the DNAbut is attached to the free 3′ end of the RNA by a template-independentRNA polymerase after transcription in the nucleus. The term “Anucleotides” or “A” refers to adenyl residues.

In a preferred embodiment, a nucleic acid molecule according to theinvention is a vector. The term “vector” is used here in its mostgeneral meaning and comprises any intermediate vehicles for a nucleicacid which, for example, enable said nucleic acid to be introduced intoprokaryotic and/or eukaryotic host cells and, where appropriate, to beintegrated into a genome. Such vectors are preferably replicated and/orexpressed in the cell. Vectors comprise plasmids, phagemids or virusgenomes. The term “plasmid”, as used herein, generally relates to aconstruct of extrachromosomal genetic material, usually a circular DNAduplex, which can replicate independently of chromosomal DNA.

According to the invention, the term “host cell” refers to any cellwhich can be transformed or transfected with an exogenous nucleic acid.The term “host cell” comprises, according to the invention, prokaryotic(e.g. E. coli) or eukaryotic cells (e.g. yeast cells and insect cells).Particular preference is given to mammalian cells such as cells fromhumans, mice, hamsters, pigs, goats, primates. The cells may be derivedfrom a multiplicity of tissue types and comprise primary cells and celllines. Specific examples include keratinocytes, peripheral bloodleukocytes, bone marrow stem cells and embryonic stem cells. In otherembodiments, the host cell is an antigen-presenting cell, in particulara dendritic cell, a monocyte or a macrophage. A nucleic acid may bepresent in the host cell in a single or in several copies and, in oneembodiment is expressed in the host cell.

According to the present invention, the term “peptide” comprises oligo-and polypeptides and refers to substances which comprise two or more,preferably 3 or more, preferably 4 or more, preferably 6 or more,preferably 8 or more, preferably 10 or more, preferably 13 or more,preferably 16 or more, preferably 20 or more, and up to preferably 50,preferably 100 or preferably 150, consecutive amino acids linked to oneanother via peptide bonds.

The term “protein” refers to large peptides, preferably peptides havingat least 151 amino acids, but the terms “peptide” and “protein” are usedherein usually as synonyms. The terms “peptide” and “protein” compriseaccording to the invention substances which contain not only amino acidcomponents but also non-amino acid components such as sugars andphosphate structures, and also comprise substances containing bonds suchas ester, thioether or disulfide bonds.

According to the present invention, a nucleic acid such as RNA mayencode a peptide or protein. Accordingly, a transcribable nucleic acidsequence or a transcript thereof may contain an open reading frame (ORF)encoding a peptide or protein. Said nucleic may express the encodedpeptide or protein. For example, said nucleic acid may be a nucleic acidencoding and expressing an antigen or a pharmaceutically active peptideor protein such as an immunologically active compound (which preferablyis not an antigen).

According to the invention, the term “nucleic acid encoding a peptide orprotein” means that the nucleic acid, if present in the appropriateenvironment, preferably within a cell, can direct the assembly of aminoacids to produce the peptide or protein during the process oftranslation. Preferably, RNA according to the invention is able tointeract with the cellular translation machinery allowing translation ofthe peptide or protein.

According to the invention, in one embodiment, RNA comprises or consistsof pharmaceutically active RNA. A “pharmaceutically active RNA” may beRNA that encodes a pharmaceutically active peptide or protein.

A “pharmaceutically active peptide or protein” has a positive oradvantageous effect on the condition or disease state of a subject whenadministered to the subject in a therapeutically effective amount.Preferably, a pharmaceutically active peptide or protein has curative orpalliative properties and may be administered to ameliorate, relieve,alleviate, reverse, delay onset of or lessen the severity of one or moresymptoms of a disease or disorder. A pharmaceutically active peptide orprotein may have prophylactic properties and may be used to delay theonset of a disease or to lessen the severity of such disease orpathological condition. The term “pharmaceutically active peptide orprotein” includes entire proteins or polypeptides, and can also refer topharmaceutically active fragments thereof. It can also includepharmaceutically active analogs of a peptide or protein. The term“pharmaceutically active peptide or protein” includes peptides andproteins that are antigens, i.e., administration of the peptide orprotein to a subject elicits an immune response in a subject which maybe therapeutic or partially or fully protective.

Examples of pharmaceutically active proteins include, but are notlimited to, cytokines and immune system proteins such as immunologicallyactive compounds (e.g., interleukins, colony stimulating factor (CSF),granulocyte colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), erythropoietin, tumor necrosisfactor (TNF), interferons, integrins, addressins, seletins, homingreceptors, T cell receptors, immunoglobulins, soluble majorhistocompatibility complex antigens, immunologically active antigenssuch as bacterial, parasitic, or viral antigens, allergens,autoantigens, antibodies), hormones (insulin, thyroid hormone,catecholamines, gonadotrophines, trophic hormones, prolactin, oxytocin,dopamine, bovine somatotropin, leptins and the like), growth hormones(e.g., human grown hormone), growth factors (e.g., epidermal growthfactor, nerve growth factor, insulin-like growth factor and the like),growth factor receptors, enzymes (tissue plasminogen activator,streptokinase, cholesterol biosynthetic or degradative, steriodogenicenzymes, kinases, phosphodiesterases, methylases, de-methylases,dehydrogenases, cellulases, proteases, lipases, phospholipases,aromatases, cytochromes, adenylate or guanylaste cyclases, neuramidasesand the like), receptors (steroid hormone receptors, peptide receptors),binding proteins (growth hormone or growth factor binding proteins andthe like), transcription and translation factors, tumor growthsuppressing proteins (e.g., proteins which inhibit angiogenesis),structural proteins (such as collagen, fibroin, fibrinogen, elastin,tubulin, actin, and myosin), blood proteins (thrombin, serum albumin,Factor VII, Factor VIII, insulin, Factor IX, Factor X, tissueplasminogen activator, protein C, von Wilebrand factor, antithrombinIII, glucocerebrosidase, erythropoietin granulocyte colony stimulatingfactor (GCSF) or modified Factor VIII, anticoagulants and the like.

In one embodiment, the pharmaceutically active protein according to theinvention is a cytokine which is involved in regulating lymphoidhomeostasis, preferably a cytokine which is involved in and preferablyinduces or enhances development, priming, expansion, differentiationand/or survival of T cells. In one embodiment, the cytokine is aninterleukin. In one embodiment, the pharmaceutically active proteinaccording to the invention is an interleukin selected from the groupconsisting of IL-2, IL-7, IL-12, IL-15, and IL-21.

The term “immunologically active compound” relates to any compoundaltering an immune response, preferably by inducing and/or suppressingmaturation of immune cells, inducing and/or suppressing cytokinebiosynthesis, and/or altering humoral immunity by stimulating antibodyproduction by B cells. Immunologically active compounds possess potentimmunostimulating activity including, but not limited to, antiviral andantitumor activity, and can also down-regulate other aspects of theimmune response, for example shifting the immune response away from aTH2 immune response, which is useful for treating a wide range of TH2mediated diseases. Immunologically active compounds can be useful asvaccine adjuvants.

If, according to the present invention, it is desired to induce orenhance an immune response by using RNA as described herein, the immuneresponse may be triggered or enhanced by the RNA. For example, proteinsor peptides encoded by the RNAs or procession products thereof may bepresented by major histocompatibility complex (MHC) proteins expressedon antigen presenting cells. The MHC peptide complex can then berecognized by immune cells such as T cells leading to their activation.

The term “disease” refers to an abnormal condition that affects the bodyof an individual. A disease is often construed as a medical conditionassociated with specific symptoms and signs. A disease may be caused byfactors originally from an external source, such as infectious disease,or it may be caused by internal dysfunctions, such as autoimmunediseases.

According to the invention, the term “disease” also refers to cancerdiseases. The terms “cancer disease” or “cancer” (medical term:malignant neoplasm) refer to a class of diseases in which a group ofcells display uncontrolled growth (division beyond the normal limits),invasion (intrusion on and destruction of adjacent tissues), andsometimes metastasis (spread to other locations in the body via lymph orblood). These three malignant properties of cancers differentiate themfrom benign tumors, which are self-limited, and do not invade ormetastasize. Most cancers form a tumor, i.e. a swelling or lesion formedby an abnormal growth of cells (called neoplastic cells or tumor cells),but some, like leukemia, do not. Examples of cancers include, but arenot limited to, carcinoma, lymphoma, blastoma, sarcoma, glioma andleukemia. More particularly, examples of such cancers include bonecancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skincancer, cancer of the head or neck, cutaneous or intraocular malignantmelanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of theanal region, stomach cancer, colon cancer, breast cancer, prostatecancer, uterine cancer, carcinoma of the sexual and reproductive organs,Hodgkin's disease, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the bladder, cancer of the kidney, renal cellcarcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), neuroectodermal cancer, spinal axis tumors,glioma, meningioma, and pituitary adenoma. The term “cancer” accordingto the invention also comprises cancer metastases.

The term “infectious disease” refers to any disease which can betransmitted from individual to individual or from organism to organism,and is caused by a microbial agent (e.g. common cold). Examples ofinfectious diseases include viral infectious diseases, such as AIDS(HIV), hepatitis A, B or C, herpes, herpes zoster (chicken-pox), Germanmeasles (rubella virus), yellow fever, dengue etc. flaviviruses,influenza viruses, hemorrhagic infectious diseases (Marburg or Ebolaviruses), and severe acute respiratory syndrome (SARS), bacterialinfectious diseases, such as Legionnaire's disease (Legionella),sexually transmitted diseases (e.g. chlamydia or gonorrhea), gastriculcer (Helicobacter), cholera (Vibrio), tuberculosis, diphtheria,infections by E. coli, Staphylococci, Salmonella or Streptococci(tetanus); infections by protozoan pathogens such as malaria, sleepingsickness, leishmaniasis; toxoplasmosis, i.e. infections by Plasmodium,Trypanosoma, Leishmania and Toxoplasma; or fungal infections, which arecaused e.g. by Cryptococcus neoformans, Histoplasma capsulatum,Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.

The term “autoimmune disease” refers to any disease in which the bodyproduces an immunogenic (i.e. immune system) response to someconstituent of its own tissue. In other words, the immune system losesits ability to recognize some tissue or system within the body as selfand targets and attacks it as if it were foreign. Autoimmune diseasescan be classified into those in which predominantly one organ isaffected (e.g. hemolytic anemia and anti-immune thyroiditis), and thosein which the autoimmune disease process is diffused through many tissues(e.g. systemic lupus erythematosus). For example, multiple sclerosis isthought to be caused by T cells attacking the sheaths that surround thenerve fibers of the brain and spinal cord. This results in loss ofcoordination, weakness, and blurred vision. Autoimmune diseases areknown in the art and include, for instance, Hashimoto's thyroiditis,Grave's disease, lupus, multiple sclerosis, rheumatic arthritis,hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus,celiac disease, Crohn's disease, colitis, diabetes, scleroderma,psoriasis, and the like.

According to the invention, an immune response may be stimulated byintroducing into a subject a suitable mRNA which codes for an antigen ora fragment thereof, e.g., a disease-associated antigen.

The term “antigen” relates to an agent comprising an epitope againstwhich an immune response is to be generated. The term “antigen” includesin particular proteins, peptides, polysaccharides, nucleic acids,especially RNA and DNA, and nucleotides. The term “antigen” alsoincludes agents, which become antigenic—and sensitizing—only throughtransformation (e.g. intermediately in the molecule or by completionwith body protein). An antigen is preferably presentable by cells of theimmune system such as antigen presenting cells like dendritic cells ormacrophages. In addition, an antigen or a processing product thereof ispreferably recognizable by a T or B cell receptor, or by animmunoglobulin molecule such as an antibody. In a preferred embodiment,the antigen is a disease-associated antigen, such as a tumor-associatedantigen, a viral antigen, or a bacterial antigen.

The term “disease-associated antigen” is used in it broadest sense torefer to any antigen associated with a disease. A disease-associatedantigen is a molecule which contains epitopes that will stimulate ahost's immune system to make a cellular antigen-specific immune responseand/or a humoral antibody response against the disease. Thedisease-associated antigen may therefore be used for therapeuticpurposes. Disease-associated antigens are preferably associated withinfection by microbes, typically microbial antigens, or associated withcancer, typically tumors.

The term “disease involving an antigen” refers to any disease whichimplicates an antigen, e.g. a disease which is characterized by thepresence of an antigen. The disease involving an antigen can be aninfectious disease, an autoimmune disease, or a cancer disease or simplycancer. As mentioned above, the antigen may be a disease-associatedantigen, such as a tumor-associated antigen, a viral antigen, or abacterial antigen.

In one embodiment, a disease-associated antigen is a tumor-associatedantigen. In this embodiment, the present invention may be useful intreating cancer or cancer metastasis. Preferably, the diseased organ ortissue is characterized by diseased cells such as cancer cellsexpressing a disease-associated antigen and/or being characterized byassociation of a disease-associated antigen with their surface.Immunization with intact or substantially intact tumor-associatedantigens or fragments thereof such as MHC class I and class II peptidesor nucleic acids, in particular mRNA, encoding such antigen or fragmentmakes it possible to elicit a MHC class I and/or a class II typeresponse and, thus, stimulate T cells such as CD8+ cytotoxic Tlymphocytes which are capable of lysing cancer cells and/or CD4+ Tcells. Such immunization may also elicit a humoral immune response (Bcell response) resulting in the production of antibodies against thetumor-associated antigen. Furthermore, antigen presenting cells (APC)such as dendritic cells (DCs) can be loaded with MHC class I-presentedpeptides by transfection with nucleic acids encoding tumor antigens invitro and administered to a patient. In one embodiment, the term“tumor-associated antigen” refers to a constituent of cancer cells whichmay be derived from the cytoplasm, the cell surface and the cellnucleus. In particular, it refers to those antigens which are produced,preferably in large quantity, intracellularly or as surface antigens ontumor cells. Examples for tumor antigens include HER2, EGFR, VEGF,CAMPATH1-antigen, CD22, CA-125, HLA-DR, Hodgkin-lymphoma or mucin-1, butare not limited thereto.

According to the present invention, a tumor-associated antigenpreferably comprises any antigen which is characteristic for tumors orcancers as well as for tumor or cancer cells with respect to type and/orexpression level. In one embodiment, the term “tumor-associated antigen”relates to proteins that are under normal conditions, i.e. in a healthysubject, specifically expressed in a limited number of organs and/ortissues or in specific developmental stages, for example, thetumor-associated antigen may be under normal conditions specificallyexpressed in stomach tissue, preferably in the gastric mucosa, inreproductive organs, e.g., in testis, in trophoblastic tissue, e.g., inplacenta, or in germ line cells, and are expressed or aberrantlyexpressed in one or more tumor or cancer tissues. In this context, “alimited number” preferably means not more than 3, more preferably notmore than 2 or 1. The tumor-associated antigens in the context of thepresent invention include, for example, differentiation antigens,preferably cell type specific differentiation antigens, i.e., proteinsthat are under normal conditions specifically expressed in a certaincell type at a certain differentiation stage, cancer/testis antigens,i.e., proteins that are under normal conditions specifically expressedin testis and sometimes in placenta, and germ line specific antigens. Inthe context of the present invention, the tumor-associated antigen ispreferably not or only rarely expressed in normal tissues or is mutatedin tumor cells. Preferably, the tumor-associated antigen or the aberrantexpression of the tumor-associated antigen identifies cancer cells. Inthe context of the present invention, the tumor-associated antigen thatis expressed by a cancer cell in a subject, e.g., a patient sufferingfrom a cancer disease, is preferably a self-protein in said subject. Inpreferred embodiments, the tumor-associated antigen in the context ofthe present invention is expressed under normal conditions specificallyin a tissue or organ that is non-essential, i.e., tissues or organswhich when damaged by the immune system do not lead to death of thesubject, or in organs or structures of the body which are not or onlyhardly accessible by the immune system. Preferably, a tumor-associatedantigen is presented in the context of MHC molecules by a cancer cell inwhich it is expressed.

Examples for differentiation antigens which ideally fulfill the criteriafor tumor-associated antigens as contemplated by the present inventionas target structures in tumor immunotherapy, in particular, in tumorvaccination are the cell surface proteins of the Claudin family, such asCLDN6 and CLDN18.2. These differentiation antigens are expressed intumors of various origins, and are particularly suited as targetstructures in connection with antibody-mediated cancer immunotherapy dueto their selective expression (no expression in a toxicity relevantnormal tissue) and localization to the plasma membrane.

Further examples for antigens that may be useful in the presentinvention are p53, ART-4, BAGE, beta-catenin/m, Bcr-abL CAMEL, CAP-1,CASP-8, CDC27/m, CDK4/m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M,ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV-E7, HPV-E6,HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1/Melan-A, MC1R,Myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190minor BCR-abL, Pm1/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 orRU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN,TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE and WT, preferably WT-1.

The term “viral antigen” refers to any viral component having antigenicproperties, i.e. being able to provoke an immune response in anindividual. The viral antigen may be a viral ribonucleoprotein or anenvelope protein.

The term “bacterial antigen” refers to any bacterial component havingantigenic properties, i.e. being able to provoke an immune response inan individual. The bacterial antigen may be derived from the cell wallor cytoplasm membrane of the bacterium.

The term “immune response”, as used herein, relates to a reaction of theimmune system such as to immunogenic organisms, such as bacteria orviruses, cells or substances. The term “immune response” includes theinnate immune response and the adaptive immune response. Preferably, theimmune response is related to an activation of immune cells, aninduction of cytokine biosynthesis and/or antibody production. It ispreferred that the immune response comprises the steps of activation ofantigen presenting cells, such as dendritic cells and/or macrophages,presentation of an antigen or fragment thereof by said antigenpresenting cells and activation of cytotoxic T cells due to thispresentation.

The term “treat” or “treatment” relates to any treatment which improvesthe health status and/or prolongs (increases) the lifespan of anindividual. Said treatment may eliminate the disease in an individual,arrest or slow the development of a disease in an individual, inhibit orslow the development of a disease in an individual, decrease thefrequency or severity of symptoms in an individual, and/or decrease therecurrence in an individual who currently has or who previously has hada disease.

In particular, the term “treatment of a disease” includes curing,shortening the duration, ameliorating, slowing down or inhibitingprogression or worsening of a disease or the symptoms thereof.

The term “immunotherapy” relates to a treatment preferably involving aspecific immune reaction and/or immune effector function(s).

The term “immunization” or “vaccination” describes the process oftreating a subject for therapeutic or prophylactic reasons.

The term “subject” or “individual”, as used herein, preferably relatesto mammals. For example, mammals in the context of the present inventionare humans, non-human primates, domesticated animals such as dogs, cats,sheep, cattle, goats, pigs, horses etc., laboratory animals such asmice, rats, rabbits, guinea pigs, etc. as well as animals in captivity,such as animals of zoos. In a preferred embodiment, the subject is ahuman.

The term “antigen presenting cell” (APC) relates to a cell of a varietyof cells capable of displaying, acquiring, and/or presenting at leastone antigen or antigenic fragment on (or at) its cell surface.Antigen-presenting cells can be distinguished in professional antigenpresenting cells and non-professional antigen presenting cells.

The term “professional antigen presenting cells” relates to antigenpresenting cells which constitutively express the MajorHistocompatibility Complex class II (MHC class II) molecules requiredfor interaction with naive T cells. If a T cell interacts with the MHCclass II molecule complex on the membrane of the antigen presentingcell, the antigen presenting cell produces a co-stimulatory moleculeinducing activation of the T cell. Professional antigen presenting cellscomprise dendritic cells and macrophages.

The term “non-professional antigen presenting cells” relates to antigenpresenting cells which do not constitutively express MHC class IImolecules, but upon stimulation by certain cytokines such asinterferon-gamma. Exemplary, non-professional antigen presenting cellsinclude fibroblasts, thymic epithelial cells, thyroid epithelial cells,glial cells, pancreatic beta cells or vascular endothelial cells.

In one embodiment of the invention, nucleic acids such as RNA areadministered to a patient by ex vivo methods, i.e. by removing cellsfrom a patient, genetically modifying said cells and reintroducing themodified cells into the patient. Transfection and transduction methodsare known to the skilled worker.

According to the invention, the term “transfection” refers tointroducing one or more nucleic acids into an organism or into a hostcell. Various methods may be employed in order to introduce according tothe invention nucleic acids into cells in vitro or in vivo. Such methodsinclude transfection of nucleic acid-CaPO₄ precipitates, transfection ofnucleic acids associated with DEAE, transfection or infection withviruses carrying the nucleic acids of interest, liposome-mediatedtransfection, and the like.

According to the invention, nucleic acids may be directed to particularcells. In such embodiments, a carrier used for administering a nucleicacid to a cell (e.g. a retrovirus or a liposome) may have a boundtargeting molecule. For example, a molecule such as an antibody specificto a surface membrane protein on the target cell, or a ligand for areceptor on the target cell may be incorporated into or bound to thenucleic acid carrier. If administration of a nucleic acid by liposomesis desired, proteins binding to a surface membrane protein associatedwith endocytosis may be incorporated into the liposome formulation inorder to enable targeting and/or absorption. Such proteins includecapsid proteins or fragments thereof which are specific to a particularcell type, antibodies to proteins that are internalized, proteinstargeting an intracellular site, and the like.

“Reporter” relates to a molecule, typically a peptide or protein, whichis encoded by a reporter gene and measured in a reporter assay.Conventional systems usually employ an enzymatic reporter and measurethe activity of said reporter.

The term “multiple cloning site” refers to a nucleic acid regioncontaining restriction enzyme sites, any one of which may be used forcleavage of, for example, a vector and insertion of a nucleic acid.

According to the invention, two elements such as nucleotides or aminoacids are consecutive, if they are directly adjacent to one another,without any interruption. For example, a sequence of x consecutivenucleotides N refers to the sequence (N)_(x).

“Restriction endonuclease” or “restriction enzyme” refers to a class ofenzymes that cleave phosphodiester bonds in both strands of a DNAmolecule within specific base sequences. They recognize specific bindingsites, referred to as recognition sequences, on a double-stranded DNAmolecule. The sites at which said phosphodiester bonds in the DNA arecleaved by said enzymes are referred to as cleavage sites. In the caseof type IIS enzymes, the cleavage site is located at a defined distancefrom the DNA binding site. According to the invention, the term“restriction endonuclease” comprises, for example, the enzymes SapI,EciI, BpiI, AarI, AloI, BaeI, BbvCI, PpiI and PsrI, BsrD1, BtsI, EarI,BmrI, BsaI, BsmBI, FauI, BbsI, BciVI, BfuAI, BspMI, BseRI, EciI, BtgZI,BpuEI, BsgI, MmeI, CspCI, BaeI, BsaMI, Mva1269I, PctI, Bse3DI, BseMI,Bst6I, Eam1104I, Ksp632I, BfiI, Bso31I, BspTNI, Eco31I, Esp3I, BfuI,Acc36I, AarI, Eco57I, Eco57MI, GsuI, AloI, Hin4I, PpiI, and PsrI.

The term “stability” of RNA relates to the “half-life” of RNA.“Half-life” relates to the period of time which is needed to eliminatehalf of the activity, amount, or number of molecules. In the context ofthe present invention, the half-life of a RNA is indicative for thestability of said RNA.

The nucleic acids such as RNA described herein, in particular when usedfor the treatments described herein, may be present in the form of apharmaceutical composition or kit comprising the nucleic acid andoptionally one or more pharmaceutically acceptable carriers, diluentsand/or excipients.

Pharmaceutical compositions are preferably sterile and contain aneffective amount of the nucleic acid.

Pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known in the art. Thepharmaceutical composition may, e.g., be in the form of a solution orsuspension.

The pharmaceutical composition may comprise salts, buffer substances,preservatives, carriers, diluents and/or excipients all of which arepreferably pharmaceutically acceptable. The term “pharmaceuticallyacceptable” refers to the non-toxicity of a material which does notinterfere with the action of the active component(s) of thepharmaceutical composition.

Salts which are not pharmaceutically acceptable may be used forpreparing pharmaceutically acceptable salts and are included in theinvention. Pharmaceutically acceptable salts of this kind comprise, in anon-limiting way, those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallyacceptable salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

Suitable buffer substances for use in the pharmaceutical compositioninclude acetic acid in a salt, citric acid in a salt, boric acid in asalt and phosphoric acid in a salt.

Suitable preservatives for use in the pharmaceutical composition includebenzalkonium chloride, chlorobutanol, paraben and thimerosal.

The term “carrier” refers to an organic or inorganic component, of anatural or non-natural (synthetic) nature, with which the activecomponent is combined in order to facilitate, enhance or enableapplication. According to the invention, the term “carrier” alsoincludes one or more compatible solid or liquid fillers, diluents orencapsulating substances, which are suitable for administration to apatient.

Possible carrier substances for parenteral administration are, e.g.,sterile water, glucose solutions, Ringer, Ringer lactate, sterile sodiumchloride solution, polyalkylene glycols, hydrogenated naphthalenes and,in particular, biocompatible lactide polymers, lactide/glycolidecopolymers or polyoxyethylene/polyoxy-propylene copolymers.

The term “excipient” when used herein is intended to indicate allsubstances which may be present in a pharmaceutical composition andwhich are not active ingredients such as, e.g., carriers, binders,lubricants, thickeners, surface active agents, preservatives,emulsifiers, buffers, flavoring agents, or colorants.

The pharmaceutical compositions described herein may be administered viaany conventional route, such as by parenteral administration includingby injection or infusion. Administration is preferably parenterally,e.g. intravenously, intraarterially, subcutaneously, in the lymph node,intradermally or intramuscularly.

Compositions suitable for parenteral administration usually comprise asterile aqueous or non-aqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer's solution and isotonicsodium chloride solution. In addition, usually sterile, fixed oils areused as solution or suspension medium.

The agents and compositions described herein are preferably administeredin effective amounts. An “effective amount” refers to the amount whichachieves a desired reaction or a desired effect alone or together withfurther doses. In the case of treatment of a particular disease or of aparticular condition, the desired reaction preferably relates toinhibition of the course of the disease. This comprises slowing down theprogress of the disease and, in particular, interrupting or reversingthe progress of the disease. The desired reaction in a treatment of adisease or of a condition may also be delay of the onset or a preventionof the onset of said disease or said condition.

An effective amount of an agent or composition described herein willdepend on the condition to be treated, the severeness of the disease,the individual parameters of the patient, including age, physiologicalcondition, size and weight, the duration of treatment, the type of anaccompanying therapy (if present), the specific route of administrationand similar factors. Accordingly, the doses administered of the agentsdescribed herein may depend on several of these parameters. In the casethat a reaction in a patient is insufficient with an initial dose,higher doses (or effectively higher doses achieved by a different, morelocalized route of administration) may be used.

The present invention is described in detail by the following figuresand examples which should be construed by way of illustration only andnot by way of limitation. On the basis of the description and theexamples, further embodiments are accessible to the skilled worker andare likewise within the scope of the invention.

FIGURES

FIGS. 1A-1B: Semi-automated screen on poly(dA:dT) stability

96 E. coli clones carrying a plasmid DNA with a poly(dA:dT)-region werepicked and inoculated in 1.4 mL in a 96 well plate for 14-16 h (37° C.,225 rpm). Bacterial culture suspensions were harvested and plasmid DNAwas purified using a Nucleospin 96 well kit (Macherey & Nagel) accordingto the manufacturer's protocol. Plasmid DNA concentration was determinedby UV spectroscopy (Nanodrop 2000, Thermo Scientific). Poly(dA:dT)integrity was determined by SacI restriction analysis (New EnglandBiolabs). The resulting fragments were resolved on an automatedcapillary gel electrophoresis (Qiagen). FIG. 1A) Example of thepoly(dA:dT) analysis of 8 clones. The bands of the internal size markerat 25 bp and 500 bp are marked with black asterisks. The expected bandsfor a poly(dA:dT) sequence of the correct length at 142 bp and 270 bpare marked with black arrows. Clone 1 and clone 4 show an additionalband resulting from a shortened poly(dA:dT) sequence, marked with redasterisks. FIG. 1B) Example of a vector map coding for a mRNA consistingof a 5′-untranslated region (5UTR), a gene of interest (GOI), the3″-untranslated region (3UTR) and the poly(A) tail (A120). The SacIrestriction sites are depicted and the lengths of the fragments uponincubation with SacI are given.

FIG. 2: Stability of different poly(dA:dT) constructs

Plasmid DNA of 96 E. coli clones of each poly(dA:dT) construct waspurified and SacI restriction analysis was performed. Construct names:A+numbers: number of adenosines 5′ of the linker sequence+L: linkersequence+number: number of adenosines 3′ of the linker sequence. Cloneswith shortened poly(dA:dT) sequence were determined and are given aspercent of the total number of E. coli clones.

FIG. 3: Stability of poly(dA:dT) constructs in different E. coli strains

E. coli strains TOP10, DH5α and XL1-blue were used for poly(dA:dT)integrity testing by SacI restriction analysis. 96 clones for theconstructs A120, A30L70 and A40L60 were tested. Number of clones withshortened poly(dA:dT) sequence are given in percent of total.

FIGS. 4A-4C: Functional in vitro characterization of differentpoly(A)-tails

The plasmids coding for the firefly luciferase gene containing eitherA120, A30L70 or A40L60 were linearized downstream of the poly(dA:dT)with a classIIS restriction enzyme thereby generating a template with noadditional nucleotide past the poly(dA:dT). Linearized plasmid DNA waspurified using carboxylated magnetic beads (Invitrogen), quantifiedspectrophotometrically and subjected to in vitro transcriptions. For invitro transcriptions T7 RNA polymerase (Fermentas), the respectivereaction buffer and 6 mM NTPs were used. For efficient capping of theRNA the GTP concentration was lowered to 1.5 mM and 6 mM of β-S-ARCA(D2)were added to the reaction and incubated for 2.5 h at 37° C. RNA waspurified via carboxylated magnetic beads (Invitrogen) and RNAconcentration and quality were assessed by spectrophotometry andanalysis on a 2100 Bionanalyzer (Agilent). FIG. 4A) 1×10⁶ human immaturedendritic cells (iDC), FIG. 4B) human fibroblasts (CCD) or FIG. 4C)murine myoblastoma cells (C2C12) cells were mixed with 10 pmol of RNArespectively and subjected to electroporation. 5×10⁴ cells were seededin X-VIVO15 media (Lonza) with additives in 24 well dishes. At 2, 4, 8,24, 48 and 72 hours after seeding firefly luciferase activities weredetermined by addition of Luciferin (Promega) in a fluorescence reader(TECAN).

FIGS. 5A-5B: Functional in vivo characterization of different poly(A)tails

BALB/c mice (n=5) were injected intravenously with RNA-lipoplexescontaining 20 μg of RNA coding for Luciferase (Luc-RNA) and carrying thedifferent poly(A)-tails A120, A30L70 or A40L60. Uptake and translationof Luc-RNA were evaluated by in vivo bioluminescence imaging using theIVIS Lumina imaging system (Caliper Life Sciences). Briefly, an aqueoussolution of D-luciferin (150 mg/kg body weight) (BD Biosciences) wasinjected i.p. 6 hours after administration of RNA lipoplexes. 5 minthereafter, emitted photons were quantified (integration time of 1 min).In vivo bioluminescence in regions of interest were quantified asaverage radiance (photons/sec/cm²/sr) using IVIS Living Image 4.0Software. The intensity of transmitted light originating from luciferaseexpressing cells within the animal was represented as a color-scaleimage, where blue is the least intense and red the most intensebioluminescence signal. Grayscale reference images of mice were obtainedunder LED low light illumination. The images were superimposed using theLiving Image 4.0 software. The luciferase signal was monitored over 48h. FIG. 5A) Luciferase activity in the spleen of the mice is shown. FIG.5B) Quantification of the cumulative luciferase signal monitored over 48hours.

FIGS. 6A-6B: Comparison of immunological response of differentpoly(A)-tails

C57BL/6 mice (n=5) were immunized intravenously in duplicates withRNA-lipoplexes containing 20 μg of RNA coding for the SIINFEKL peptide(SEQ ID NO: 1) carrying the different poly A tails A120, A30L70 orA40L60 on days 0 and 3. The frequencies of antigen specific CD8⁺ T cellswere determined in peripheral blood via SIINFEKL-MHC (SEQ ID NO: 1)tetramer staining 5 days after the last immunization (Day 8). Briefly,hypotonicly lysed blood samples were incubated at 4° C. with anti-CD8antibody (Invitrogen) and H-2 K^(b)/SIINFEKL (SEQ ID NO: 1) tetramer(Beckman-Coulter) and washed to remove unbound antibodies prior to theflow cytometry analysis. Flow cytometric data were acquired on aFACS-Calibur analytical flow cytometer and analyzed by using FlowJo(Tree Star) software. RNA profile was obtained from the 2100 Bioanalyzerof RNA coding for luciferase carrying poly(A)-tails A40L60, A30L70 andA120 respectively. FIG. 6A Gating strategy for antigen-specific CD8⁺ Tcells. FIG. 6B Frequencies of antigen-specific CD8⁺ T cells in CD8⁺ Tcells.

EXAMPLES Example 1: Semi-Automated Screen on Poly(dA:dT) Stability

A semi-automated process was established to screen a large number of E.coli clones for the integrity of the critical poly(dA:dT) sequenceregion encoded on the plasmid carried by individual E. coli clones. Forscreening of one specific poly(dA:dT) construct, 96 E. coli clones wereinoculated and incubated in a 96 well plate at 37° C. Cells wereharvested by centrifugation and plasmids were purified on a 96 wellplate vacuum-based purification platform. The tested plamid DNAscontained three SacI restriction sites, cleaving the vector twice in the3-UTR (3″-untranslated region) and once downstream of the poly(dA:dT)sequence. SacI restriction resulted always in 2 specific bands of 142 bpand 270 bp in size which allowed the calculation of the length of thepoly(dA:dT). The third band represented the vector backbone and theantigen with a size depending on the inserted antigen (GOI=gene ofinterest). An exemplary vector map with the position of the restrictionsites and the lengths of the fragments is depicted in FIG. 1B.

To monitor a large number of clones, samples of the SacI restrictiondigest were applied on a semi-automated capillary electrophoresis systemand band patterns between 25 bp and 500 bp were analyzed in highresolution (FIG. 1A shows an example of a restriction analysis of 8clones). The bands of the internal size standard at 25 bp and 500 bp aremarked with black asterisks (*). The bands which represent an intactpoly(dA:dT) at 142 bp and 270 bp are marked with black arrows (->).Clone 1 and clone 4 show a subpopulation with a shortened poly(dA:dT)region which results in an additional band between 142 bp and 270 bp(marked with red asterisks (*)). Instability of the poly(dA:dT) is givenas the ratio of clones with shortened poly(dA:dT) sequence to cloneswith an intact poly(dA:dT) sequence.

Example 2: Stability Testing of Different Poly(dA:dT) Constructs

As a model antigen the SIINFEKL peptide (SEQ ID NO: 1) was chosenbecause in previous experiments the poly(dA:dT) instability of thisantigen was reproducibly determined between 50-60% and providestherefore a large experimental window for stability testing. 10different poly(dA:dT) constructs were designed and fused directly behindthe SIINFEKL peptide (SEQ ID NO: 1). A 10 nucleotide linker (L) wasinserted in the poly(dA:dT) stretch in different positions of thepoly(dA:dT) sequence. The linker sequence (GCATATGACT (SEQ ID NO: 2))was chosen in a way to contain a balanced contribution of all 4nucleotides (2×G, 2×C, 3×T and 3×A). 4 constructs were designed with thelinker in the middle of the poly(dA:dT) starting with 45 adenosineresidues (45×A) on each side (A45L45) with a step-wise increase of 5×Aboth sides ending with 60×A on each side of the linker (A50L50, A55L55and A60L60, respectively). The 6 remaining constructs contained likeA50L50 100× A in total. However, the linker was inserted after 20×A,followed by the linker sequence and another 80×A (A20L80). Accordingly,the linker was inserted after 30×A (A30L70), 40×A (A40L60), 60×A(A60L40), 70×A (A70L30) and 80×A (A80L20) respectively. 96 clones ofeach of the 10 constructs were analyzed for poly(dA:dT) integrity withthe described restriction analysis method. All 10 linker containingconstructs showed a beneficial effect on poly(dA:dT) stability comparedto the A120 (see FIG. 2). The determined stability data is summarized inTable 1. Construct A45L45 showed a more than 6-fold higher stabilitycompared to the control A120, however the step-wise increase of thetotal length of the poly(dA:dT) led to a higher instability as reflectedby only 1.66-fold remaining stabilization of A60L60. Stabilization ofconstructs with 100×A and the linker sequence at varying positions ofthe poly(dA:dT) sequence ranged from 2.9-fold for A20L80 to 13-fold forA40L60. Surprisingly, A30L70 and A40L60 showed a particular highstabilization of the poly(dA:dT) region. Taken together, our resultsdemonstrate that the insertion of a 10 nucleotide random sequence has astabilizing effect on the poly(dA:dT) integrity. Especially the regionbetween position 30 and position 50 of the poly(dA:dT) region isparticular sensitive to poly(dA:dT) shortening. Introduction of linkersequences in this sequence area led to a further increases of thepoly(dA:dT) stability by at least 2-fold as compared to the otherconstructs (see Table 1 and FIG. 2).

TABLE 1 Summary of Poly(dA:dT) stability testing. Depicted is thepercentage of clones with shortened poly(dA:dT) sequence and theresulting stabilization of the poly(dA:dT) sequence compared to thepolyA120. Poly(dA:dT) Cleavage Stablization construct [% of testedclones] [fold of A120] A120 55.9 1 A45L45 8.8 6.4 A50L50 10.7 5.2 A55L5521.1 2.7 A60L60 33.7 1.7 A60L40 8.9 6.3 A70L30 13.8 4.0 A80L20 13.6 4.1A40L60 4.3 13.0 A30L70 4.4 12.7 A20L80 19.3 2.9

Example 3: Stability of Poly(dA:dT) Constructs in Different E. coliStrains

In further experiments the specificity and functionality of the superiorstability of the constructs A30L70 and A40L60 was tested. Thepossibility that the observed results of the stability testing arerestricted to the tested E. coli strain TOP10 was evaluated by includingtwo other E. coli strains in the testing. Testing for A30L70 and A40L60was repeated with DH5a, XL1-blue and TOP10 as control respectively.These strains were chosen as i) having a high genetic diversity (seeTable 2) and ii) representing E. coli strains which are widely used inmolecular biology laboratories.

Instability of the A120 was measured for DH5α at 42% and for XL1-blue at61.8% and was therefore considered to be comparable to the instabilitydetected for E. coli TOP10 strain (see FIG. 3). Both, A30L70 and A40L60showed an instability between 3-4%, only for A40L60 in TOP10 instabilitywas slightly elevated to 6.8%. Testing 3 different laboratory strains ofE. coli confirmed the results on poly(dA:dT) stabilization. Theintroduction of a 10 nucleotide linker sequence in the cleavagesensitive region at position 30-50 was identified as a general principlefor the genetic stabilization of poly(dA:dT) sequences in different E.coli strains.

TABLE 2 Genotypes of the tested E. coli strains Strain Genotype TOP10F-, mcrA, Δ(mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, nupG, recA1,araD139, Δ(ara-leu)7697, galK galU rpsL(Str^(R)), endA1, λ- DH5α F-,endA1, glnV44, thi-1, recA1, relA1, gyrA96, deoR, nupG, Φ80, lacZΔM15,Δ(lacZYA-argF)U169, hsdR17(r_(K) ⁻ m_(K) ⁺), λ- XL1- endA1, gyrA96(nal^(R)), thi-1, recA1, relA1, lac, blue glnV44, F′[::Tn10, proAB⁺,lacI^(q), Δ(lacZ)M15], hsdR17(r_(K) ⁻ m_(K) ⁺)

Example 4: Functional In Vitro Characterization of DifferentPoly(A)-Tails

Luciferase reporter-based experiments were performed to elucidate theimpact of the identified stabilized poly(A)-tails A30L70 and A40L60 onthe functionality of the RNA molecules. The constructs A30L70, A40L60and A120 were fused to a firefly luciferase reporter gene and therespective messenger RNA was generated by in vitro transcription. TheRNA molecules showed comparable integrity and were used for cellelectroporation (see Table 3). RNAs were electroporated into humanimmature dendritic cells, isolated from human bloods which represent thetarget cells for the company's mRNA tumor vaccine approach. Luciferasetranslation was monitored over a period of 72 hours. The 3 differentmRNA molecules were equally expressed with only minor differences (FIG.4A). To prove that the functionality of mRNAs in general is notinfluenced by the nature of the poly(A)-tails, the experiment wasrepeated in a human fibroblast cell line (CCD cells) and a murinemyoblast cell line (C2C12) (FIGS. 4B and 4C). Although a celltype-specific pattern of the mRNA translation was monitored over thetime neither human nor murine cell lines showed differences in proteinexpression by mRNAs containing different poly(A)-tails.

TABLE 3 Integrity of luciferase encoding IVT RNAs RNA Integrity [%]hAg-Kozak-Luciferase-2hBgUTR-A40L60 79hAg-Kozak-Luciferase-2hBgUTR-A30L70 81 hAg-Kozak-Luciferase-2hBgUTR-A12083

These results demonstrate that the chosen poly(A)-tails have only minorimpact on total mRNA functionality in vitro. Therefore linker sequenceinsertions into the poly(dA:dT) region at position 30 and position 40,respectively, allow a substantial genetic stabilization by maintainingfull functionality of the respective RNA molecules.

Example 5: Functional In Vivo Characterization of DifferentPoly(A)-Tails

For the systemic in vivo application of mRNA for vaccination, RNAlipoplexes are generated by formulation of the RNA together with lipidsand administered intravenously. The RNA lipoplexes are meant to targetthe spleen and to be taken up by immature dendritic cells whichtranslate the respective mRNA. It was aimed to test the two stabilizedpoly(A)-tails, i.e. A30L70 and A40L60 in a mouse experiment to ensurefunctional protein expression in vivo. RNA with A120 served as anexpression control. Three groups of BALB/c mice with 5 animals each wereinjected intravenously with RNA-lipoplexes containing firefly luciferaseencoding RNAs which had been used for the functional in vitro testing(Table 3) with the different poly(A)-tails (A30L70, A40L60 and A120).Firefly luciferase expression was monitored over 48 hours using an invivo bioluminescence imaging system (FIG. 5A). The quantification of thecumulative luciferase signals is shown in FIG. 5B. Neither location northe intensity of the luciferase signal differed significantly betweenthe RNAs with different poly(A)-tails proofing that both stabilizedpoly(A)-tails are suitable for systemic in vivo applications.

Example 6: Immunological Response to Different Poly(A)-Tails

In a last set of experiments it was assessed if the stabilizedpoly(A)-tails, A30L70 and A40L60 have an influence on the specificimmune response induced by the mRNA vaccine. The stabilizedpoly(A)-tails and the control A120 were therefore fused to the SIINFEKLpeptide (SEQ ID NO: 1) as for the stability testing before. The 3 RNAscontaining the poly(A)-tails A30L70, A40L60 and A120 were generated byin vitro transcription and showed comparable quality and integrity(Table 4). 3 groups of C57/BL6 mice, two times 5 animals each, wereinjected intravenously in duplicates on day 0 and day 3 with RNAlipoplexes containing the different SIINFEKL (SEQ ID NO: 1) RNAs. TheRNA-induced immune response was analyzed by determining the frequency ofantigen-specific CD8⁺ T cells 5 days after the last immunization (day 8)by SIINFEKL-MHC (SEQ ID NO: 1) tetramer staining. The respective gatingstrategy by FACS analysis is depicted in FIG. 6A. The comparison ofantigen-specific CD8⁺ T cell frequencies showed that the RNAs with alltested poly(A)-tails induced an immune response. Thereby no significantdifferences were detected neither within the same group (2×5 animals foreach RNA) nor between the 3 groups which received the different IVT RNAsdemonstrating that the stabilized poly(A)-tails did not influence thespecific immune response induced by the mRNA (FIG. 6B).

TABLE 4 Integrity of SIINFEKL (SEQ ID NO: 1) enconding IVT RNAs  IVT RNAIntegrity [%] hAg-Kozak-sec-SIINFEKL(SEQ ID NO: 1)- 82 MITD-2hBgUTR-A40L60  hAg-Kozak-sec-SIINFEKL(SEQ ID NO: 1)- 81 MITD-2hBgUTR-A30L70  hAg-Kozak-sec-SIINFEKL(SEQ ID NO: 1)- 83 MITD-2hBgUTR-A120 

By establishing a restriction-based analysis method we can show herethat the poly(dA:dT) region coding for the poly(A)-tail of an mRNA isgenetically instable in common E. coli strains. This instability leadsto labor-intensive screening efforts in order to obtain clones with astable poly(dA:dT) sequence. We demonstrated that the insertion of a 10nucleotide linker sequence stabilizes this sequence stretch. Therebyposition 30 to 50 have been identified as being in especially sensitiveto poly(dA:dT) shortening. Linker insertions in this particular regionincreased the stability further by at least factor 2 compared toinsertions at other positions. Stability testing was confirmed inseveral commonly used E. coli strains. The sequence insertions did notalter the functionality of the respective in vitro transcribed RNAs asdemonstrated in several cell lines and by comparison of in vivo activityin mice. Last, the RNA-induced immune response was not influenced by themodification of the poly(A)-tail. Taken together, we identified a toolto stabilize the poly(dA:dT) region genetically which facilitateshandling with the respective plasmid DNA and thereby neither influencingthe RNA in vitro and in vivo functionality nor the induction of anRNA-specific immune response.

1.-25. (canceled)
 26. RNA, which is obtainable by in vitro transcriptionusing, as a template, a nucleic acid molecule comprising in the 5′→3′direction of transcription: (a) a promoter; (b) a transcribable nucleicacid sequence or a nucleic acid sequence for introducing a transcribablenucleic acid sequence; and (c) a nucleic acid sequence which, whentranscribed under the control of the promoter (a), codes for a modifiedpolyadenyl sequence of at least 80 consecutive nucleotides, wherein themodified polyadenyl sequence comprises: a linker sequence comprising atleast one T, C, or G nucleotide; a first sequence of at least 20 Aconsecutive nucleotides, which is 5′ of the linker sequence; and asecond sequence of at least 20 A consecutive nucleotides, which is 3′ ofthe linker sequence.
 27. The RNA of claim 26, wherein the linkersequence is a sequence of 2 or more consecutive nucleotides, wherein thefirst and the last nucleotide of said sequence of 2 or more consecutivenucleotides is a nucleotide selected from the group consisting of T, C,and G.
 28. The RNA of claim 26, wherein the modified polyadenyl sequenceof at least 80 consecutive nucleotides comprises at least 90nucleotides.
 29. The RNA of claim 26, wherein the linker sequence islocated within a region from position 21 to position 80 of said modifiedpolyadenyl sequence.
 30. The RNA of claim 27, wherein the linkersequence has a length of at least 3 nucleotides.
 31. The RNA of claim27, wherein said sequence of 2 or more consecutive nucleotides comprises3 or fewer consecutive A nucleotides.
 32. The RNA of claim 26, whereinthe nucleic acid sequences (b) and (c) under the control of the promoter(a) can be transcribed to give a common transcript.
 33. The RNA of claim26, wherein the modified polyadenyl sequence of at least 80 consecutivenucleotides is located at the 3′ end of a transcript.
 34. The RNA ofclaim 26, wherein the nucleic acid molecule is a closed circularmolecule or a linear molecule.
 35. The RNA of claim 26, wherein thenucleic acid molecule comprises the transcribable nucleic acid sequence,which comprises a nucleic acid sequence coding for a peptide or protein.36. The RNA of claim 26, wherein the nucleic acid molecule furthercomprises one or more members selected from the group consisting of: (i)a reporter gene; (ii) a selectable marker; and (iii) an origin ofreplication.
 37. The RNA of claim 26, wherein the RNA is mRNA.
 38. TheRNA of claim 26, wherein the nucleic acid molecule comprises the nucleicacid sequence for introducing a transcribable nucleic acid sequence,which comprises a multiple cloning site.
 39. Use of the RNA of claim 26for transfecting a host cell.
 40. The use of claim 39, wherein the hostcell is an antigen-presenting cell.
 41. The use of claim 40, wherein theantigen-presenting cell is a dendritic cell, a monocyte or a macrophage.42. Use of the RNA of claim 26 for vaccination.
 43. A nucleic acidmolecule comprising in the 5′ →3′ direction of transcription: (a) apromoter; (b) a transcribable nucleic acid sequence or a nucleic acidsequence for introducing a transcribable nucleic acid sequence; and (c)a nucleic acid sequence which, when transcribed under the control of thepromoter (a), codes for a modified polyadenyl sequence of at least 80consecutive nucleotides, wherein the modified polyadenyl sequencecomprises: a linker sequence comprising at least one T, C, or Gnucleotide; a first sequence of at least 20 A consecutive nucleotides,which is 5′ of the linker sequence; and a second sequence of at least 20A consecutive nucleotides, which is 3′ of the linker sequence.